Separating agent including polysaccharide derivative having a polycyclic structure

ABSTRACT

The present invention provides a separating agent for enantiomeric isomers, which has an excellent ability to identify asymmetry. That is, the present invention relates to a separating agent for enantiomeric isomers which includes as an effective component a polysaccharide derivative having a bicyclic or more structure having an aromatic, alicyclic, or heterocyclic ring.

REFERENCE TO RELATED APPLICATIONS

This is a division of Ser. No. 10/467,201, filed Aug. 5, 2003, which wasthe national stage of International Application No. PCT/JP02/04160,filed Apr. 25, 2002, which International Application was not publishedin English.

TECHNICAL FIELD TO WHICH THE INVENTION BELONGS

The present invention relates to a separating agent including as aneffective component a polysaccharide derivative having a bicyclic ormore structure containing an aromatic ring, an alicyclic ring, or aheterocyclic ring (hereinafter referred to as a polycyclic structure)and to an enantiomeric isomer separating agent that is used forseparation of various chemical substances, in particular, for opticalresolution.

PRIOR ART

Many organic compounds have enantiomeric isomers that have the samephysical and chemical properties as one another, for example, boilingpoint, melting point and solubility, but are different from one anotherin physiological properties. This is because proteins and glucides bythemselves that constitute an organism in most cases are composed of oneenantiomeric isomer and there arises a difference in the manner ofaction on the other enantiomeric isomer and thus a difference inphysiological activity arises. In particular, in the field of medicine,it is often the case that enantiomeric isomers have a significantdifference in efficacy or toxicity. For this reason, the Ministry ofHealth, Labour and Welfare of Japan prescribes in the Guideline for theProduction of Medicines that “in the case where the drug is a racemicform, it is desirable that the dynamic behaviors of absorption,distribution, metabolism and excretion be made on each isomer”.

As described above, since physical properties and chemical properties,for example, the boiling point, melting point and solubility, of theenantiomeric isomers are quite the same, they cannot be analyzed by theordinary separation means. Accordingly, studies on the technology ofanalyzing a wide variety of enantiomeric isomers simply and with highprecision have been intensively made. As an analytical technique inresponse to these requirements, an optical separation method by highperformance liquid chromatography (HPLC), in particular an opticalseparation method by a chiral column for HPLC has been advanced. Thechiral column as used herein is an asymmetry identifying agent by itselfor a chiral immobilizing phase including an asymmetry identifying agentused as carried on a suitable carrier.

For example, optically active poly(triphenylmethyl methacrylate) (JP57-150432 A), cellulose, amylose derivative (Y. Okamoto, M. Kawashimaand K. Hatada, J. Am. Chem. Soc., 106, 5337, 1984), and ovomucoid (JP63-307829 A), which is a protein, have been developed. Among the manychiral immobilizing phases for HPLC, optical resolution columns thatcarry cellulose or an amylose derivative on silica gel are known to havehigh asymmetry identifying ability for a very wide variety of compounds.In recent years, studies have been advanced on preparative liquidchromatography for an optically active substance on an industrial scaleby using a chiral immobilizing phase for HPLC and a simulated moving bedchromatography in combination (Phram Tech Japan, 12, 43), and not onlyto fully separate but also to increase the productivity of preparativechromatography, a chiral immobilizing phase has been demanded thatperforms further improved separation of the target compound ofpreparative separation, that is, has a greater value of separationcoefficient α.

Also, recently, associated with the orientation in the art toward microanalysis technologies, enantiomeric isomer separation thin layerchromatography (chiral TLC) that is capable of performing the separationof enantiomeric isomers by a separation operation simpler thanenantiomeric isomer separation in the field of capillary electrophoresis(CE) or HPLC has received high attention. Accordingly, application ofpolysaccharide derivatives having high asymmetry identifying ability tothese fields of the art has been demanded.

DISCLOSURE OF THE INVENTION

The inventors of the present invention have made extensive research witha view to solving the above-mentioned problems, and as a result theyhave found that a polysaccharide derivative having a sterically bulkypolycyclic structure has excellent asymmetry identifying ability,thereby achieving the present invention.

Therefore, the present invention relates to a separating agent forenantiomeric isomers including as an effective component apolysaccharide derivative having a polycyclic structure.

The present invention provides use of a polysaccharide derivativecontaining a bicyclic or more structure (hereinafter referred to as apolycyclic structure) containing an aromatic ring, an alicyclic ring ora heterocyclic ring as a separating agent for enantiomeric isomers and amethod of separating enantiomeric isomers with a polysaccharidederivative containing a bicyclic or more structure (hereinafter referredto a polycyclic structure) containing an aromatic ring, an alicyclicring or a heterocyclic ring.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, description will be made of embodiments of the presentinvention in detail.

As a polysaccharide that constitutes a polysaccharide derivative havinga polycyclic structure used in the present invention, any syntheticpolysaccharide, any natural polysaccharide, and any modified naturalpolysaccharide may be used so long as they have an optical activity.Those which have a high regularity for the binding form are moredesired. There are exemplified β-1,4-glucan (cellulose), α-1,4-glucan(amylose, amylopectin), α-1,6-glucan (dextran), β-1,6-glucan (busturan),β-1,3-glucan (for example, cardran, schizophyllan, etc.), α-1,3-glucan,β-1,2-glucan (Crown Gall polysaccharide), β-1,4-galactan, β-1,4-mannan,α-1,6-mannan, β-1,2-fructan (inulin), β-2,6-fructan (levan),β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan, α-1,4-N-acetylchitosan(chitin), pullulan, agarose, alginic acid, and the like. Also, thepolysaccharide includes starch containing amylose. Among those,cellulose, amylose, β-1,4-xylan, β-1,4-chitosan, chitin, β-1,4-mannan,inulin, and cardran, which are readily available as the polysaccharidehaving high purity, are preferred. Cellulose and amylose areparticularly preferred.

These polysaccharides have a number average degree of polymerization (anaverage number of pyranose rings or furanose rings contained in onemolecule) of 5 or more, preferably 10 or more and desirably 1,000 orless in consideration of ease of handling although there is noparticular upper limit.

In the present invention, polysaccharide derivatives refer to compoundsincluding a polysaccharide with a part or all of the hydroxyl groupsthereof bonded to a compound having a functional group reactive with thehydroxyl group or groups through an ester bond, an urethane bond or anether bond, with a carbamate derivative or an ester derivative thereofbeing preferable. Particularly preferred polysaccharide derivatives usedin the present invention are ester derivatives or carbamate derivativesof polysaccharides having 0.1 or more ester bond or urethane bond per 1glucose unit and the carbamate derivatives are more preferably used.

In the present invention, the polycyclic structure means a bicyclic ormore structure containing an aromatic ring, an alicyclic ring or aheterocyclic ring, with a structure containing an aromatic ring beingpreferable. The number of rings bonded is not particularly limited but abicyclic ring is preferable. Specific examples of the group having apolycyclic structure include a fluorenyl group, an indanyl group, ananthryl group, a pyrenyl group, a phenanthryl group, a quinolyl group, apentarenyl group, an indenyl group, a naphthyl group, an azurenyl group,and a heptanyl group.

The polysaccharide derivative having a polycyclic structure used in thepresent invention can be obtained by reacting a polysaccharide with acompound having a polycyclic structure and a functional group reactivewith a hydroxyl group of the polysaccharide to form an ester bond, anurethane bond, an ether bond or the like. The compound having apolycyclic structure and a functional group reactive with a hydroxylgroup of the polysaccharide includes those compounds that have afunctional group reactive with the above-mentioned hydroxyl group andfurther have the above-mentioned polycyclic structure, for example,9H-furorenyl isocyanate and 5-indanyl isocyanate.

The polysaccharide derivative having a polycyclic structure of thepresent invention is a substance that is an extremely useful substanceas a functional material and is useful as an enantiomeric isomerseparating agent, in particular, a chiral immobilizing phase forchromatography. To use the polysaccharide derivative of the presentinvention as a separating agent in the separation of compounds orenantiomeric isomers, generally it is used in chromatographic methodssuch as a gas chromatographic method, a liquid chromatographic method, athin layer chromatographic method, a supercritical chromatographicmethod, a capillary electrophoretic method, and a continuous liquidpreparative chromatographic method. In addition, it is also possible toperform membrane separation by carrying it on a membrane.

As the chiral immobilizing phase for chromatography using thepolysaccharide derivative of the present invention, an immobilizingphase for liquid chromatography, an immobilizing phase for thin layerchromatography, an asymmetry identifying agent added to anelectrophoretic solution in capillary electrophoresis represented by amicelle electroconductive chromatographic method, and immobilizing phasefor preparative continuous liquid chromatography represented by asimulated moving bed chromatography are preferable.

To apply the separating agent of the present invention to a liquidchromatography method, there is a method for filling the separatingagent as a powder in a column, a method of coating it on a capillarycolumn, a method of forming a capillary with the separating agent andutilizing the inner wall thereof, and so on. Generally, the separatingagent is converted to a powder. To convert the separating agent into apowder, it is preferred that the separating agent be pulverized or madeinto the form of beads. The size of the particles may vary depending onthe size of the column to be used, and the particle size is preferably 1μm to 10 mm, and more preferably 1 μm to 300 μm. The particles arepreferably porous.

Further, in order to improve the pressure resistance property of theseparating agent, prevent swelling or contraction of the separatingagent by substitution of solvents, and increase the theoretical numberof stages, it is preferred that the separating agent be held on acarrier. The size of the carrier may vary depending on the size of thecolumn or plate to be used; generally the size is 1 μm to 10 mm, andmore preferably 1 μm to 300 μm. The carrier is preferably porous and hasan average pore diameter of preferably 10 Å to 100 μm, and morepreferably 50 Å to 50,000 Å. The carrying amount of the separating agentis 1 to 100% by weight, preferably 5 to 50% by weight based on thecarrier.

The method of carrying the polysaccharide derivative to the carrier maybe either a chemical method or a physical method. As the chemicalmethod, there is a method in which part of hydroxyl groups are protectedat the time of forming derivative of the polysaccharide and after thederivative is formed, deprotection is performed and the deprotectedhydroxyl group and silica gel are chemically bonded (Y. Okamoto et al.,J. Liq. Chromatogr., 10 (8&9), 1613, 1987). As the physical method,there is a method in which the polysaccharide derivative is dissolved ina solvent in which the polysaccharide derivative is soluble and renderedwell mixed with the carrier and the solvent is distilled off under areduced pressure, under warming or under an air stream or the like.

As the carrier, mention may be made of a porous organic carrier and aporous inorganic carrier. The porous inorganic carrier is preferable.Suitable examples of the porous organic carrier include polymersubstances such as polystyrene, polyacrylamide, and polyacrylate.Suitable examples of the porous inorganic carrier include synthetic ornatural substances such as silica, alumina, magnesia, titanium oxide,glass, silicates, and kaolin. To improve its affinity for thepolysaccharide derivative, the carrier may be subjected to a surfacetreatment. The method of the surface treatment includes a silanetreatment with an organosilane compound or a surface treatment withplasma polymerization, and so on.

EFFECT OF THE INVENTION

According to the present invention, an enantiomeric isomer separatingagent having a high asymmetry identifying ability can be provided.

EXAMPLES

The present invention will be described in detail by examples. However,the present invention should not be considered as being limited to theseexamples.

Example 1

[1] Synthesis of cellulose tris(9H-fluorenyl carbamate) (1a)

After 0.30 g of cellulose (trade name: Avicell, manufactured by MerckCo.) and 0.21 g of lithium chloride were dried for 3 hours, 2.0 ml ofdimethylacetamide (DMA) was added thereto and allowed to swell at 90° C.to 100° C. for one night. Thereafter, 6.0 ml of pyridine was added and1.3 equivalents of 9H-fluorenyl isocyanate was added and allowed toreact for 6 hours. A carbamate derivative was deposited byreprecipitation from the reaction mixture and filtered through a glassfilter, and then dried under vacuum to obtain 1.16 g of cellulosetris(9H-fluorenyl carbamate) represented by the formula (1a). Theelemental analysis values of the obtained (1a) are shown in Table 1.

[2] Fabrication of a filler by carrying (1a) on silica gel

0.75 g of the carbamate derivative (1a) obtained in [1] was dissolved in10 ml of tetrahydrofuran (THF) and the solution was perfused uniformlyon 3 g of silica gel (manufactured by Daiso Co., Ltd., particle size of7 μm, thin-pore diameter of 1,000 Å), and then the solvent was distilledoff to fabricate a filler on which cellulose tris(9H-fluorenylcarbamate) (1a) was carried.

[3] Fabrication of a column filled with a filler by carrying (1a) onsilica gel

2.5 g of the carried type filler fabricated in [2] was pressed andfilled into a stainless steel-made column of φ 0.46 cm×L 25 cm by aslurry filling method to fabricate a separation column for enantiomericisomers.

Example 2

[1] Synthesis of cellulose tris(5-indanyl carbamate) (1b)

After 0.20 g of cellulose (trade name: Avicell, manufactured by MerckCo.) and 0.15 g of lithium chloride were dried for 3 hours, 1.5 ml ofDMA was added thereto and allowed to swell at 90° C. to 100° C. for onenight. Thereafter, 5.0 ml of pyridine was added and 1.5 equivalents of5-indanyl isocyanate was added and allowed to react for 6 hours. Acarbamate derivative was deposited by reprecipitation from the reactionmixture and filtered through a glass filter, and then dried under vacuumto obtain 0.53 g of cellulose tris(5-indanyl carbamate) represented bythe formula (1b). The elemental analysis values of the obtained (1b) areshown in Table 1.

[2] Fabrication of a filler by carrying (1b) on silica gel

The carbamate derivative (1b) obtained in [1] was used to fabricate afiller on which cellulose tris(5-indanyl carbamate) (1b) was carried inthe same manner as in [2] of Example 1.

[3] Fabrication of a column filled with a filler by carrying (1b) onsilica gel

The carried type filler fabricated in [2] was filled into a stainlesssteel-made column of 100 0.20 cm×L 25 cm in the same manner as in [3] ofExample 1 to fabricate a separation column for enantiomeric isomers.

Example 3

[1] Synthesis of amylose tris(5-indanyl carbamate) (2b)

After 0.20 g of amylose (trade name: AS-50, manufactured by Ajinoki Co.,Ltd.) and 0.15 g of lithium chloride were dried for 3 hours, 1.5 ml ofDMA was added thereto and allowed to swell at 90° C. to 100° C. for onenight. Thereafter, 6.0 ml of pyridine was added and 1.6 equivalents of5-indanyl isocyanate was added and allowed to react for 6 hours. Acarbamate derivative was deposited by reprecipitation from the reactionmixture and filtered through a glass filter, and then dried under vacuumto obtain 0.69 g of amylose tris(5-indanyl carbamate) represented by theformula (2b). The elemental analysis values of the obtained (2b) areshown in Table 1.

[2] Fabrication of a filler by carrying (2b) on silica gel

The carbamate derivative (2b) obtained in [1] was used to fabricate afiller on which amylose tris(5-indanyl carbamate) (2b) was carried inthe same manner as in [2] of Example 1.

[3] Fabrication of a column filled with a filler by carrying (2b) onsilica gel

The carried type filler fabricated in [2] was used and filled into astainless steel-made column of φ 0.20 cm×L 25 cm in the same manner asin [3] of Example 1 to fabricate a separation column for enantiomericisomers. TABLE 1 Elemental analysis result C % H % N % (1a) Calculatedvalue 73.55 4.76 5.36 Analysis value 71.95 4.98 5.13 (1b) Calculatedvalue 67.59 5.83 6.57 Analysis value 65.49 5.75 6.26 (2b) Calculatedvalue 67.59 5.83 6.57 Analysis value 66.19 5.76 6.44

Comparative Example 1

By using cellulose trisphenyl carbamate as a separating agent, anenantiomeric isomer separation column was fabricated by the same methodas that in Example 1 in JP 60-108751 A.

Application Example 1

By using the columns fabricated in Examples 1 to 3 and ComparativeExample 1, optical resolution of racemic forms (a) to (g) describedbelow was performed by a liquid chromatographic method under theconditions described below. The results are shown in Table 2.

<Analysis Conditions>

Moving phase: Hexane/isopropanol=90/10 (v/v)

Flow rate: 0.5 ml/min for the columns of Example 1 and ComparativeExample 1 and 0.1 ml/min for the columns of

Examples 2 and 3

Temperature: 25° C.

Detection: 254 nm

Note that the separation coefficient (α) in the table is defined asfollows.α=k ₂ ′/k ₁′

Here, k₁′ is the holding coefficient of a relatively weakly heldenantiomeric isomer, and k₂′ is the holding coefficient of a relativelystrongly held enantiomeric isomer. TABLE 2 Target racemic Separationcoefficient (α) form for Comparative separation Example 1 Example 2Example 3 Example 1 (a) 1.56 1.41 1.46 1.19 (b) to 1 1.25 1.49 1.0  (c)1.26 1.78 1.30 1.47 (d) to 1 1.21 1.41 to 1 (e) to 1 1.5 to 1 1.0  (f)1.14 1.67 to 1 1.52 (g) to 1 1.57 1.21 to 1

1. A separating agent for enantiomeric isomers comprising as aneffective component a polysaccharide derivative that contains apolycyclic structure containing an aromatic ring, an alicyclic ring or aheterocyclic ring, with the proviso that said polysaccharide derivativeis not amylose tris(5-indanylcarbamate).
 2. The separating agentaccording to claim 1, wherein the polysaccharide derivative having apolycyclic structure is a cellulose derivative or an amylose derivative.3. The separating agent according to claim 1, wherein the polysaccharidederivative having a polycyclic structure contains an aromatic ring. 4.The separating agent according to claim 1, wherein the polysaccharidederivative having a polycyclic structure is an ester derivative or acarbamate derivative.
 5. The separating agent according to claim 1,wherein the separating agent is used as a chiral immobilizing phase forchromatography.
 6. The separating agent according to claim 5, whereinthe chiral immobilizing phase for chromatography is an immobilizingphase for liquid chromatography.
 7. The separating agent according toclaim 5, wherein the chiral immobilizing phase for chromatography is animmobilizing phase for a thin layer chromatography.
 8. The separatingagent according to claim 5, wherein the chiral immobilizing phase forchromatography is an asymmetry identifying agent added to anelectrophoretic solution in a capillary electrophoresis.
 9. Theseparating agent according to claim 5, wherein the chiral immobilizingphase for chromatography is an immobilizing phase for a preparativecontinuous liquid chromatography.
 10. A separating agent forenantiomeric isomers comprising a polysaccharide derivative containingat least one member selected from the group consisting of a fluorenylgroup, an indanyl group, an anthryl group, a pyrenyl group, aphenanthryl group, a quinolyl group, a pentarenyl group, an indenylgroup, a naphthyl group, an azurenyl group and a heptanyl group.
 11. Theseparating agent according to claim 10, wherein the polysaccharidederivative is a cellulose carbamate derivative.
 12. The separating agentaccording to claim 10, wherein the polysaccharide derivative is anamylose carbamate derivative.
 13. The separating agent according toclaim 11, wherein the cellulose carbamate derivative is cellulosetris(9H-fluorenylcarbamate).